Bilayer conformation of fusion peptide of influenza virus hemagglutinin: a molecular dynamics simulation study

Unraveling the conformation of membrane-bound viral fusion peptides is essential for understanding how those peptides destabilize the bilayer topology of lipids that is important for virus-cell membrane fusion. Here, molecular dynamics (MD) simulations were performed to investigate the conformation of the 20 amino acids long fusion peptide of influenza hemagglutinin of strain X31 bound to a dimyristoyl phosphatidylcholine (DMPC) bilayer. The simulations revealed that the peptide adopts a kinked conformation, in agreement with the NMR structures of a related peptide in detergent micelles. The peptide is located at the amphipathic interface between the headgroups and hydrocarbon chains of the lipid by an energetically favorable arrangement: The hydrophobic side chains of the peptides are embedded into the hydrophobic region and the hydrophilic side chains are in the headgroup region. The N-terminus of the peptide is localized close to the amphipathic interface. The molecular dynamics simulations also revealed that the peptide affects the surrounding bilayer structure. The average hydrophobic thickness of the lipid phase close to the N-terminus is reduced in comparison with the average hydrophobic thickness of a pure dimyristoyl phosphatidylcholine bilayer.     

Molecular Dynamics Simulations of Homo-oligomeric Bundles Embedded Within a Lipid Bilayer

Using molecular dynamics simulations, we studied the structure, interhelix interactions, and dynamics of transmembrane proteins. Specifically, we investigated homooligomeric helical bundle systems consisting of synthetic α-helices with either the sequence Ac-(LSLLLSL)₃-NH₂ (LS2) or Ac-(LSSLLSL)₃-NH₂ (LS3). The LS2 and LS3 helical peptides are designed to have amphipathic characteristics that form ion channels in membrane. We simulated bundles containing one to six peptides that were embedded in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer and placed between two lamellae of water. We aim to provide a fundamental understanding of how amphipathic helical peptides interact with each other and their dynamical behaviors in different homooligomeric states. To understand structural properties, we examined the helix lengths, tilt angles of individual helices and the entire bundle, interhelix distances, interhelix cross-angles, helix hydrophobic-to-hydrophilic vector projections, and the average number of interhelix hydrophilic (serine–serine) contacts lining the pore of the transmembrane channel. To analyze dynamical properties, we calculated the rotational autocorrelation function of each helix and the cross-correlation of the rotational velocity between adjacent helices. The observed structural and dynamical characteristics show that higher order bundles containing four to six peptides are composed of multiple lower order bundles of one to three peptides. For example, the LS2 channel was found to be stable in a tetrameric bundle composed of a “dimer of dimers.” In addition, we observed that there is a minimum of two strong hydrophilic contacts between a pair of adjacent helices in the dimer to tetramer systems and only one strong hydrophilic interhelix contact in helix pairs of the pentamer and hexamer systems. We believe these results are general and can be applied to more complex ion channels, providing insight into ion channel stability and assembly.     

Molecular Dynamics Simulations of Homo-oligomeric Bundles Embedded Within a Lipid Bilayer

Using molecular dynamics simulations, we studied the structure, interhelix interactions, and dynamics of transmembrane proteins. Specifically, we investigated homooligomeric helical bundle systems consisting of synthetic α-helices with either the sequence Ac-(LSLLLSL)₃-NH₂ (LS2) or Ac-(LSSLLSL)₃-NH₂ (LS3). The LS2 and LS3 helical peptides are designed to have amphipathic characteristics that form ion channels in membrane. We simulated bundles containing one to six peptides that were embedded in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer and placed between two lamellae of water. We aim to provide a fundamental understanding of how amphipathic helical peptides interact with each other and their dynamical behaviors in different homooligomeric states. To understand structural properties, we examined the helix lengths, tilt angles of individual helices and the entire bundle, interhelix distances, interhelix cross-angles, helix hydrophobic-to-hydrophilic vector projections, and the average number of interhelix hydrophilic (serine–serine) contacts lining the pore of the transmembrane channel. To analyze dynamical properties, we calculated the rotational autocorrelation function of each helix and the cross-correlation of the rotational velocity between adjacent helices. The observed structural and dynamical characteristics show that higher order bundles containing four to six peptides are composed of multiple lower order bundles of one to three peptides. For example, the LS2 channel was found to be stable in a tetrameric bundle composed of a “dimer of dimers.” In addition, we observed that there is a minimum of two strong hydrophilic contacts between a pair of adjacent helices in the dimer to tetramer systems and only one strong hydrophilic interhelix contact in helix pairs of the pentamer and hexamer systems. We believe these results are general and can be applied to more complex ion channels, providing insight into ion channel stability and assembly.     

Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography.

Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.     

Exploring models of the influenza A M2 channel: MD simulations in a phospholipid bilayer

The M2 protein of influenza A virus forms homotetrameric helix bundles, which function as proton-selective channels. The native form of the protein is 97 residues long, although peptides representing the transmembrane section display ion channel activity, which (like the native channel) is blocked by the antiviral drug amantadine. As a small ion channel, M2 may provide useful insights into more complex channel systems. Models of tetrameric bundles of helices containing either 18 or 22 residues have been simulated while embedded in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine bilayer. Several different starting models have been used. These suggest that the simulation results, at least on a nanosecond time scale, are sensitive to the exact starting structure. Electrostatics calculations carried out on a ring of four ionizable aspartate residues at the N-terminal mouth of the channel suggest that at any one time, only one will be in a charged state. Helix bundle models were mostly stable over the duration of the simulation, and their helices remained tilted relative to the bilayer normal. The M2 helix bundles form closed channels that undergo breathing motions, alternating between a tetramer and a dimer-of-dimers structure. Under these conditions either the channel forms a pocket of trapped waters or it contains a column of waters broken predominantly at the C-terminal mouth of the pore. These waters exhibit restricted motion in the pore and are effectively "frozen" in a way similar to those seen in previous simulations of a proton channel formed by a four-helix bundle of a synthetic leucine-serine peptide (, Biophys. J. 77:2400-2410).     

Peptide Aggregation and Pore Formation in a Lipid Bilayer: A Combined Coarse-Grained and All Atom Molecular Dynamics Study

We present a simulation study where different resolutions, namely coarse-grained (CG) and all-atom (AA) molecular dynamics simulations, are used sequentially to combine the long timescale reachable by CG simulations with the high resolution of AA simulations, to describe the complete processes of peptide aggregation and pore formation by alamethicin peptides in a hydrated lipid bilayer. In the 1-μs CG simulations the peptides spontaneously aggregate in the lipid bilayer and exhibit occasional transitions between the membrane-spanning and the surface-bound configurations. One of the CG systems at t = 1 μs is reverted to an AA representation and subjected to AA simulation for 50 ns, during which water molecules penetrate the lipid bilayer through interactions with the peptide aggregates, and the membrane starts leaking water. During the AA simulation significant deviations from the α-helical structure of the peptides are observed, however, the size and arrangement of the clusters are not affected within the studied time frame. Solid-state NMR experiments designed to match closely the setup used in the molecular dynamics simulations provide strong support for our finding that alamethicin peptides adopt a diverse set of configurations in a lipid bilayer, which is in sharp contrast to the prevailing view of alamethicin oligomers formed by perfectly aligned helical alamethicin peptides in a lipid bilayer.     

Surface binding of alamethicin stabilizes its helical structure: molecular dynamics simulations.

Alamethicin is an amphipathic alpha-helical peptide that forms ion channels. An early event in channel formation is believed to be the binding of alamethicin to the surface of a lipid bilayer. Molecular dynamics simulations are used to compare the structural and dynamic properties of alamethicin in water and alamethicin bound to the surface of a phosphatidylcholine bilayer. The bilayer surface simulation corresponded to a loosely bound alamethicin molecule that interacted with lipid headgroups but did not penetrate the hydrophobic core of the bilayer. Both simulations started with the peptide molecule in an alpha-helical conformation and lasted 2 ns. In water, the helix started to unfold after approximately 300 ps and by the end of the simulation only the N-terminal region of the peptide remained alpha-helical and the molecule had collapsed into a more compact form. At the surface of the bilayer, loss of helicity was restricted to the C-terminal third of the molecule and the rod-shaped structure of the peptide was retained. In the surface simulation about 10% of the peptide/water H-bonds were replaced by peptide/lipid H-bonds. These simulations suggest that some degree of stabilization of an amphipathic alpha-helix occurs at a bilayer surface even without interactions between hydrophobic side chains and the acyl chain core of the bilayer.     

Phospholemman transmembrane structure reveals potential interactions with Na+/K+-ATPase

Phospholemman (PLM) is a 72-residue bitopic cardiac transmembrane protein, which acts as a modulator of the Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger and possibly forms taurine channels in nonheart tissue. This work presents a high resolution structural model obtained from a combination of site-specific infrared spectroscopy and experimentally constrained high throughput molecular dynamics (MD) simulations. Altogether, 37 experimental constraints, including nine long range orientational constraints, have been used during MD simulations in an explicit lipid bilayer/water system. The resulting tetrameric alpha-helical bundle has an average helix tilt of 7.3 degrees and a crossing angle close to 0 degrees . It does not reveal a hydrophilic pore, but instead strong interactions between various residues occlude any pore. The helix-helix packing is unusual, with Gly(19) and Gly(20) pointing to the outside of the helical bundle, facilitating potential interaction with other transmembrane proteins, thus providing a structural basis for the modulatory effect of PLM on the Na(+)/K(+)-ATPase. A two-stage model of interaction between PLM and the Na(+)/K(+)-ATPase is discussed involving PLM-ATPase interaction and subsequent formation of an unstable PLM trimer, which readily interacts with surrounding ATPase molecules. Further unconstrained MD simulations identified other packing models of PLM, one of which could potentially undergo a conformational transition to an open pore.     

Biophysical investigations of membrane perturbations by polypeptides using solid-state NMR spectroscopy (review)

Polypeptides have been prepared by solid-phase peptide synthesis and labelled with 15N at single sites to be used for static or magic angle spinning solid-state NMR spectroscopy. After reconstitution into oriented membranes, the alignment of polypeptide alpha-helices with respect to the bilayer surface is accessible by proton-decoupled 15N solid-state NMR spectroscopy. In addition, limiting values of rotational diffusion coefficients are obtained. The effects of membrane inserted peptides on the bilayer phospholipids have been investigated by 2H and 31P solid-state NMR spectroscopy. Long hydrophobic peptides such as the channel-forming domains of Vpu of HIV-1 or M2 of influenza A adopt stable alignments approximately parallel to the bilayer normal in agreement with models suggesting transmembrane helical bundle formation. The 15N chemical shift data agree with tilt angles of approximately 20 degrees and 33 degrees, respectively. In contrast, multi-charged amphipathic alpha-helices adopt stable orientations parallel to the bilayer surface. In the presence of these peptides, decreased order parameters of the fatty acyl chains, membrane thinning, and the loss of long-range order are observed. Peptides that change topology in a pH dependent manner are more potent in antibiotic assays under experimental conditions where they show in-plane alignments. This result suggests that their detergent-like properties, rather than the formation of transmembrane helical bundles, are responsible for their cell-killing activities. Topological equilibria are also observed within proteins or for polypeptides that do not match the hydrophobic thickness of the bilayer.     

Molecular dynamics methods to predict peptide locations in membranes: LAH4 as a stringent test case

Determining the structure of membrane-active peptides inside lipid bilayers is essential to understand their mechanism of action. Molecular dynamics simulations can easily provide atomistic details, but need experimental validation. We assessed the reliability of self-assembling (or “minimum-bias”) and potential of mean force (PMF) approaches, using all-atom (AA) and coarse-grained (CG) force-fields. The LAH4 peptide was selected as a stringent test case, since it is known to attain different orientations depending on the protonation state of its four histidine residues.In all simulations the histidine side-chains inserted in the membrane when neutral, while they interacted with phospholipid headgroups in their charged state. This led to transmembrane orientations for neutral-His LAH4 in all minimum-bias AA simulations and in most CG trajectories. By contrast, the charged-His peptide stabilized membrane defects in AA simulations, whereas it was located at the membrane surface in some CG trajectories, and interacted with both lipid leaflets in others. This behavior is consistent with the higher antimicrobial activity and membrane-permeabilizing behavior of the charged-His LAH4. In addition, good agreement with solid-state NMR orientational data was observed in AA simulations.PMF calculations correctly predicted a higher membrane affinity for the neutral-His peptide. Interestingly, the structures and relative populations of PMF local free-energy minima corresponded to those determined in the less computationally demanding minimum-bias simulations.These data provide an indication about the possible membrane-perturbation mechanism of the charged-His LAH4 peptide: by interacting with lipid headgroups of both leaflets through its cationic side-chains, it could favor membrane defects and facilitate translocation across the bilayer.     

Hydrophilic surface maps of channel-forming peptides: analysis of amphipathic helices

Ion channels may be formed by bundles of amphipathic -helices aligned parallel to one another and spanning a lipid bilayer membrane, with the hydrophilic faces of the helices lining a central pore. In order to provide insight into the packing of such helices in bundles, a method has been developed to evalute hydrophilic surface maps of amphipathic -helices and to display these surfaces in a readily interpretable form. The procedure is based upon empirical energy calculations of interactions of a water molecule with an amphipathic -helix. The method has been applied to three channel-forming peptides: Staphylococcal -toxin; alamethicin; and a synthetic leucine- and serine-containing peptide. Particular emphasis is placed upon the effects of sidechain conformational flexibility on hydrophilic surface maps. Å family of models of the -toxin; helix is generated by a simulated annealing procedure. The results of hydrophilic surface map analyses provide more exact definition of the centre of the hydrophilic face of amphipathic helices, and of the variation of the position of the centre in response to changes in sidechain conformation. This information is used to define families of preliminary models for a given ion channel, as is illustrated for -toxin.Abbreviations ABNR adopted basis Newton-Raphson - CFP channel-forming peptide - HSM hydrophilic surface map - MHP molecular hydrophobicity potential - RMSD root mean square deviation Correspondence to: M. S. P. Sansom     

Model generation of viral channel forming 2B protein bundles from polio and coxsackie viruses

2B is a 99 amino acid membrane protein encoded by enteroviruses such as polio and coxsackie viruses with two transmembrane domains. The protein is found to make membranes of infected cells permeable. Using a computational approach which positions the models and assesses stability by molecular dynamics (MD) simulations a putative tetrameric bundle model of 2B is generated. The bundles show a pore lining motif of three lysines followed by a serine. The bundle is discussed in terms of different possible orientations of the helices in the membrane and the consequences this has on the in vivo activity of 2B.     

Membrane insertion topology of the central apolipoprotein A-I region. Fluorescence studies using single tryptophan mutants

Apolipoprotein A-I (apoAI) contains several amphipathic α-helices. To carry out its function, it exchanges between lipid-free and different lipidated states as bound to membranes or to lipoprotein complexes of different morphology, size, and composition. When bound to membranes or to spherical lipoprotein surfaces, it is thought that most α-helices arrange with their long axis parallel to the membrane surface. However, we previously found that a central region spanning residues 87-112 is exclusively labeled by photoactivable reagents deeply located into the membrane (Co?rsico et al. (2001) J. Biol. Chem. 276, 16978-16985). A pair of amphipathic α-helical repeats with a particular charge distribution is predicted in this region. In order to study their insertion topology, three single tryptophan mutants, each one containing the tryptophan residue at a selected position in the hydrophobic face of the central Y-helices (W@93, W@104, and W@108), were used. From the accessibility to quenchers located at different membrane depths, distances from the bilayer center of 13.4, 10.5, and 15.7 ? were estimated for positions 93, 104, and 108, respectively. Reported data also indicate that distances between homologous positions (in particular for W@93 and W@104) are very short in dimers in aqueous solution, but they are larger in membrane-bound dimers. Data indicate that an intermolecular central Y-helix bundle would penetrate the membrane perpendicularly to the membrane surface. Intermolecular helix-helix interactions would occur through the hydrophilic helix faces in the membrane-bound bundle but through the hydrophobic faces in the case of dimers in solution.     

The interaction of phospholipase A2 with a phospholipid bilayer: coarse-grained molecular dynamics simulations

A number of membrane-active enzymes act in a complex environment formed by the interface between a lipid bilayer and bulk water. Although x-ray diffraction studies yield structures of isolated enzyme molecules, a detailed characterization of their interactions with the interface requires a measure of how deeply such a membrane-associated protein penetrates into a lipid bilayer. Here, we apply coarse-grained (CG) molecular dynamics (MD) simulations to probe the interaction of porcine pancreatic phospholipase A2 (PLA2) with a lipid bilayer containing palmitoyl-oleoyl-phosphatidyl choline and palmitoyl-oleoyl-phosphatidyl glycerol molecules. We also used a configuration from a CG-MD trajectory to initiate two atomistic (AT) MD simulations. The results of the CG and AT simulations are evaluated by comparison with available experimental data. The membrane-binding surface of PLA2 consists of a patch of hydrophobic residues surrounded by polar and basic residues. We show this proposed footprint interacts preferentially with the anionic headgroups of the palmitoyl-oleoyl-phosphatidyl glycerol molecules. Thus, both electrostatic and hydrophobic interactions determine the location of PLA2 relative to the bilayer. From a general perspective, this study demonstrates that CG-MD simulations may be used to reveal the orientation and location of a membrane-surface-bound protein relative to a lipid bilayer, which may subsequently be refined by AT-MD simulations to probe more detailed interactions.     

Molecular dynamics investigation of membrane-bound bundles of the channel-forming transmembrane domain of viral protein U from the human immunodeficiency virus HIV-1

Molecular dynamics (MD) simulations have been carried out on bundles of the channel-forming transmembrane (TM) domain of the viral protein U (VPU(1-27) and VPU(6-27)) from the human immunodeficiency virus (HIV-1). Simulations of hexameric and pentameric bundles of VPU(6-27) in an octane/water membrane mimetic system suggested that the pentamer is the preferred oligomer. Accordingly, an unconstrained pentameric helix bundle of VPU(1-27) was then placed in a hydrated palmitoyl-oleyl-3-n-glycero-phosphatidylethanolamine (POPE) lipid bilayer and its structural properties calculated from a 3-ns MD run. Some water molecules, initially inside the channel lumen, were expelled halfway through the simulation and the bundle adopted a conical structure reminiscent of previous MD results obtained for VPU(6-27) in an octane/water system. The pore constriction generated may correspond to a closed state of the channel and underlies the relocation of the W residue toward the pore lumen. The relative positions of the helices with respect to the bilayer and their interactions with the lipids are discussed. The observed structure is stabilized via specific interactions between the VPU helices and the carbonyl oxygen atoms of the lipid molecules, particularly at the Q and S residues.     

The design, synthesis, and crystallization of an alpha-helical peptide

Twelve- and sixteen-residue peptides have been designed to form tetrameric alpha-helical bundles. Both peptides are capable of folding into amphiphilic alpha-helices, with leucyl residues along one face and glutamyl and lysyl residues along the opposite face. Four such amphiphilic alpha-helices are capable of forming a noncovalently bonded tetramer. Neighboring helices run in antiparallel directions in the design, so that the complex has 222 symmetry. In the designed tetramer, the leucyl side chains interdigitate in the center in a hydrophobic interaction, and charged side chains are exposed to the solvent. The designed 12-mer (ALPHA-1) has been synthesized, and it forms helical aggregates in aqueous solution as judged by circular dichroic spectroscopy. It has also been crystallized and characterized by x-ray diffraction. The crystal symmetry is compatible with (but does not prove) the design. The design can be extended to a four-alpha-helical bundle formed from a single polypeptide by adding three peptide linkers.